restriction enzyme paci  (New England Biolabs)


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    New England Biolabs restriction enzyme paci
    Impact of H1.2 KxUb variants on chromatosome assembly. a Schematic overview of 12-mer chromatosome array formation containing H1.2 or H1.2 KxUbs. b EMSA of different arrays analyzed; crDNA indicates competitor DNA. c Overview of MNase digestion assay which probes DNA accessibility of <t>nucleosome</t> and chromatosome arrays (left). The arrays were treated with MNase for indicated times, digested by proteinase K, and analyzed by agarose gel electrophoresis. Undigested arrays were quantified and plotted over time (middle). Shown are mean values +/− standard deviation, n = 3. For statistical analysis, the area under the curve for the digestion of the different arrays was determined and analyzed (right); shown are mean values +/− standard deviation, n = 3, one-way ANOVA with Tukey’s multiple comparisons test with α = 0.05, 95% confidence interval, exact p values with p ≤ 0.05 are shown, ns (not significant) p > 0.05. d <t>PacI</t> digestion assay. PacI cleaves the array site-specifically within the linker DNA between the repetitive nucleosome positioning sequences resulting in the formation of mono-nucleosomes and mono-chromatosomes.
    Restriction Enzyme Paci, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    restriction enzyme paci - by Bioz Stars, 2022-08
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    1) Product Images from "Site-specific ubiquitylation acts as a regulator of linker histone H1"

    Article Title: Site-specific ubiquitylation acts as a regulator of linker histone H1

    Journal: Nature Communications

    doi: 10.1038/s41467-021-23636-5

    Impact of H1.2 KxUb variants on chromatosome assembly. a Schematic overview of 12-mer chromatosome array formation containing H1.2 or H1.2 KxUbs. b EMSA of different arrays analyzed; crDNA indicates competitor DNA. c Overview of MNase digestion assay which probes DNA accessibility of nucleosome and chromatosome arrays (left). The arrays were treated with MNase for indicated times, digested by proteinase K, and analyzed by agarose gel electrophoresis. Undigested arrays were quantified and plotted over time (middle). Shown are mean values +/− standard deviation, n = 3. For statistical analysis, the area under the curve for the digestion of the different arrays was determined and analyzed (right); shown are mean values +/− standard deviation, n = 3, one-way ANOVA with Tukey’s multiple comparisons test with α = 0.05, 95% confidence interval, exact p values with p ≤ 0.05 are shown, ns (not significant) p > 0.05. d PacI digestion assay. PacI cleaves the array site-specifically within the linker DNA between the repetitive nucleosome positioning sequences resulting in the formation of mono-nucleosomes and mono-chromatosomes.
    Figure Legend Snippet: Impact of H1.2 KxUb variants on chromatosome assembly. a Schematic overview of 12-mer chromatosome array formation containing H1.2 or H1.2 KxUbs. b EMSA of different arrays analyzed; crDNA indicates competitor DNA. c Overview of MNase digestion assay which probes DNA accessibility of nucleosome and chromatosome arrays (left). The arrays were treated with MNase for indicated times, digested by proteinase K, and analyzed by agarose gel electrophoresis. Undigested arrays were quantified and plotted over time (middle). Shown are mean values +/− standard deviation, n = 3. For statistical analysis, the area under the curve for the digestion of the different arrays was determined and analyzed (right); shown are mean values +/− standard deviation, n = 3, one-way ANOVA with Tukey’s multiple comparisons test with α = 0.05, 95% confidence interval, exact p values with p ≤ 0.05 are shown, ns (not significant) p > 0.05. d PacI digestion assay. PacI cleaves the array site-specifically within the linker DNA between the repetitive nucleosome positioning sequences resulting in the formation of mono-nucleosomes and mono-chromatosomes.

    Techniques Used: Agarose Gel Electrophoresis, Standard Deviation

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    New England Biolabs restriction enzyme paci
    Impact of H1.2 KxUb variants on chromatosome assembly. a Schematic overview of 12-mer chromatosome array formation containing H1.2 or H1.2 KxUbs. b EMSA of different arrays analyzed; crDNA indicates competitor DNA. c Overview of MNase digestion assay which probes DNA accessibility of <t>nucleosome</t> and chromatosome arrays (left). The arrays were treated with MNase for indicated times, digested by proteinase K, and analyzed by agarose gel electrophoresis. Undigested arrays were quantified and plotted over time (middle). Shown are mean values +/− standard deviation, n = 3. For statistical analysis, the area under the curve for the digestion of the different arrays was determined and analyzed (right); shown are mean values +/− standard deviation, n = 3, one-way ANOVA with Tukey’s multiple comparisons test with α = 0.05, 95% confidence interval, exact p values with p ≤ 0.05 are shown, ns (not significant) p > 0.05. d <t>PacI</t> digestion assay. PacI cleaves the array site-specifically within the linker DNA between the repetitive nucleosome positioning sequences resulting in the formation of mono-nucleosomes and mono-chromatosomes.
    Restriction Enzyme Paci, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction enzyme paci/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    restriction enzyme paci - by Bioz Stars, 2022-08
    95/100 stars
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    Impact of H1.2 KxUb variants on chromatosome assembly. a Schematic overview of 12-mer chromatosome array formation containing H1.2 or H1.2 KxUbs. b EMSA of different arrays analyzed; crDNA indicates competitor DNA. c Overview of MNase digestion assay which probes DNA accessibility of nucleosome and chromatosome arrays (left). The arrays were treated with MNase for indicated times, digested by proteinase K, and analyzed by agarose gel electrophoresis. Undigested arrays were quantified and plotted over time (middle). Shown are mean values +/− standard deviation, n = 3. For statistical analysis, the area under the curve for the digestion of the different arrays was determined and analyzed (right); shown are mean values +/− standard deviation, n = 3, one-way ANOVA with Tukey’s multiple comparisons test with α = 0.05, 95% confidence interval, exact p values with p ≤ 0.05 are shown, ns (not significant) p > 0.05. d PacI digestion assay. PacI cleaves the array site-specifically within the linker DNA between the repetitive nucleosome positioning sequences resulting in the formation of mono-nucleosomes and mono-chromatosomes.

    Journal: Nature Communications

    Article Title: Site-specific ubiquitylation acts as a regulator of linker histone H1

    doi: 10.1038/s41467-021-23636-5

    Figure Lengend Snippet: Impact of H1.2 KxUb variants on chromatosome assembly. a Schematic overview of 12-mer chromatosome array formation containing H1.2 or H1.2 KxUbs. b EMSA of different arrays analyzed; crDNA indicates competitor DNA. c Overview of MNase digestion assay which probes DNA accessibility of nucleosome and chromatosome arrays (left). The arrays were treated with MNase for indicated times, digested by proteinase K, and analyzed by agarose gel electrophoresis. Undigested arrays were quantified and plotted over time (middle). Shown are mean values +/− standard deviation, n = 3. For statistical analysis, the area under the curve for the digestion of the different arrays was determined and analyzed (right); shown are mean values +/− standard deviation, n = 3, one-way ANOVA with Tukey’s multiple comparisons test with α = 0.05, 95% confidence interval, exact p values with p ≤ 0.05 are shown, ns (not significant) p > 0.05. d PacI digestion assay. PacI cleaves the array site-specifically within the linker DNA between the repetitive nucleosome positioning sequences resulting in the formation of mono-nucleosomes and mono-chromatosomes.

    Article Snippet: Analysis of nucleosome and chromatosome arrays PacI digest : To analyze saturation of the nucleosome array and evaluate H1.2-binding to the nucleosome, 79 fmol arrays were digested with 5 U of the restriction enzyme PacI (NEB) in 10 mM HEPES/KOH (pH 7.6), 50 mM KCl, 1.5 mM MgCl2, 0.5 mM EGTA for 1 h at 37 °C.

    Techniques: Agarose Gel Electrophoresis, Standard Deviation

    Symmetric barcoding and amplification of chromatin fragments A) Barcode adapters (top) are 64 bp double-stranded oligonucleotides with universal primers, barcode sequences and restriction sites, whose symmetric design allows ligation on either side. Schematic (bottom left) depicts possible outcomes of ligation in drops, including symmetrically labeled nucleosomes, asymmetrically labeled nucleosomes, and adapter concatemers. Concatemers are removed by digestion of PacI sites formed by adapter juxtaposition (bottom center), allowing selective PCR amplification of symmetrically adapted chromatin fragments (bottom right). See also Supplementary Figure 2 . B) Gel electrophoresis for DNA products at successive assay stages: left : DNA ladder; MNase : DNA fragments purified after capture, lysis and MNase digestion of single cells in drops confirm efficient digestion to mononucleosomes (∼1 million drops collected); Concat : Illumina library prepared from adaptor-ligated chromatin fragments without PacI digestion reveals overwhelming concatemer bias. Library : Illumina library prepared from adaptor-ligated chromatin fragments digested with PacI, reveals appropriate MNase digestion pattern, shifted by the size of barcode and Illumina adapters. C) Pie charts depict numbers of uniquely aligned sequencing read that satisfy successive filtering criteria (values reflect data from 100 single cells, averaged over 82 trials). We select reads that have barcode sequences on both ends (top) with matching sequence (middle). We then apply a Poisson model to identify barcodes that represent single cells (bottom). D) Heatmap depicts homogeneity of barcode selection. Barcodes (rows) are colored according to their relative prevalence (rank order) across 37 experiments (columns). The absence of bias towards particular barcodes (light or dark horizontal stripes) indicates the homogeneity of the barcode library. The mean normalized rank over all barcodes (right) is close to 0.5, consistent with balanced representation. E) Stability of the barcode library emulsion over time. The fraction of reads with matching barcodes on both ends is plotted as a function of time from encapsulation of the barcode library. F) The microfluidics system was applied to barcode a mixed suspension of human and mouse cells. For each barcode, plot depicts the number of reads aligning to the mouse genome (y-axis) versus the number of reads aligning to the human genome (x-axis). The data suggest that a vast majority of barcodes is unique to a single cell.

    Journal: Nature biotechnology

    Article Title: Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state

    doi: 10.1038/nbt.3383

    Figure Lengend Snippet: Symmetric barcoding and amplification of chromatin fragments A) Barcode adapters (top) are 64 bp double-stranded oligonucleotides with universal primers, barcode sequences and restriction sites, whose symmetric design allows ligation on either side. Schematic (bottom left) depicts possible outcomes of ligation in drops, including symmetrically labeled nucleosomes, asymmetrically labeled nucleosomes, and adapter concatemers. Concatemers are removed by digestion of PacI sites formed by adapter juxtaposition (bottom center), allowing selective PCR amplification of symmetrically adapted chromatin fragments (bottom right). See also Supplementary Figure 2 . B) Gel electrophoresis for DNA products at successive assay stages: left : DNA ladder; MNase : DNA fragments purified after capture, lysis and MNase digestion of single cells in drops confirm efficient digestion to mononucleosomes (∼1 million drops collected); Concat : Illumina library prepared from adaptor-ligated chromatin fragments without PacI digestion reveals overwhelming concatemer bias. Library : Illumina library prepared from adaptor-ligated chromatin fragments digested with PacI, reveals appropriate MNase digestion pattern, shifted by the size of barcode and Illumina adapters. C) Pie charts depict numbers of uniquely aligned sequencing read that satisfy successive filtering criteria (values reflect data from 100 single cells, averaged over 82 trials). We select reads that have barcode sequences on both ends (top) with matching sequence (middle). We then apply a Poisson model to identify barcodes that represent single cells (bottom). D) Heatmap depicts homogeneity of barcode selection. Barcodes (rows) are colored according to their relative prevalence (rank order) across 37 experiments (columns). The absence of bias towards particular barcodes (light or dark horizontal stripes) indicates the homogeneity of the barcode library. The mean normalized rank over all barcodes (right) is close to 0.5, consistent with balanced representation. E) Stability of the barcode library emulsion over time. The fraction of reads with matching barcodes on both ends is plotted as a function of time from encapsulation of the barcode library. F) The microfluidics system was applied to barcode a mixed suspension of human and mouse cells. For each barcode, plot depicts the number of reads aligning to the mouse genome (y-axis) versus the number of reads aligning to the human genome (x-axis). The data suggest that a vast majority of barcodes is unique to a single cell.

    Article Snippet: Library preparation To minimize the abundance of barcode adaptors concatemers we add 1uL of PacI restriction enzyme (R0547L, NEB, USA) and 2.5ul of NEB Buffer 1 to each sample of 100 cells in 21.5uL of TE and incubate at 37C for 2 hours and then at 65C for 20 minutes.

    Techniques: Amplification, Ligation, Labeling, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Purification, Lysis, Sequencing, Selection